Enzymatic reaction monitoring method based on optical bar code

Abstract

The invention discloses an enzymatic reaction monitoring method based on an optical bar code, and belongs to the technical field of optical fiber microcavity sensing and biological sensing. The inner surface of a microbubble cavity is coated with a polymer sensitive to pH, and when the polymer is exposed in a specific enzymatic reaction solution, the polymer can be combined with hydrogen ions generated after a reaction of a solution to be detected, so that the refractive index of the polymer is changed; then an optical bar code database is established, corresponding optical bar code information and the pH value are extracted from the database according to the detected enzymatic reaction spectrum, and pH value monitoring of the enzymatic reaction solution to be detected is completed. According to the method, the pH value of the specific acid solution can be conveniently and efficiently detected, and biological information such as the pH value can be intuitively monitored by utilizing an optical barcode technology in an echo wall mode. The method can extract additional information related to the biosensing process from the overall behavior of the WGM spectrum, and can be widely applied to the fields of biochemical reaction detection, environmental protection and the like.

Description

Technical Field

[0001] The invention relates to the fields of optical fiber microcavity sensing and biosensing, and in particular to a method for measuring pH value of an enzymatic reaction integrated with a whispering gallery mode microbubble cavity and an optical barcode display method. Background Art

[0002] Biosensors based on optical microcavities, including Fabry-Perot (FP), photonic crystals, and whispering gallery modes (WGM), are classified according to their light wave confinement mechanisms and have been widely used to monitor proteins, lipids, viruses, and surfactants on modified interfaces. Light waves are able to circulate millions of times within optical resonators, significantly enhancing the interaction between light and probe particles, thereby improving sensing sensitivity. Among various microcavities, whispering gallery mode microresonators stand out due to their small mode volume, narrow linewidth, and high quality factor (Q factor), which greatly facilitates the detection of subtle changes in analytes during tracking of the resonance wavelength. WGM resonances are affected by externally induced changes in the cavity refractive index and physical dimensions, and various WGM resonators have been demonstrated to monitor biochemical reactions in contact with surfaces by tracking changes in the resonance wavelength. However, the overall monitoring of multimodal spectra has always been a technical challenge in the field of analytical spectroscopy. Summary of the invention

[0003] In order to solve the coupling problem between the whispering gallery mode microbubble cavity and the tapered optical fiber, a microbubble encapsulation structure was designed, which can stably encapsulate the microbubble cavity and stably couple with the tapered optical fiber to produce the whispering gallery phenomenon. The high Q value and high sensitivity of the microbubble cavity and the idea of ​​monitoring pH value are used to monitor the reaction process of the enzymatic reaction, and the optical barcode technology is used to intuitively display the spectral characteristics of the reaction results, including the conversion of the resonant wavelength, mode spacing, line width, and coupling depth into optical barcodes and lines of width, position, and color, and a reference barcode database corresponding to pH value is established.

[0004] To achieve the above object, the present invention provides the following scheme: a method for monitoring an enzymatic reaction based on an optical barcode, comprising the following steps:

[0005] S1. introducing the liquid to be tested into the microbubble cavity coated with poly (N-isopropylacrylamide); coupling the light in the tapered optical fiber into the poly (N-isopropylacrylamide) layer on the inner wall of the microbubble cavity in the form of an evanescent field, causing total reflection to generate a whispering gallery mode;

[0006] S2. Collect multiple spectra with pH values ​​from 5 to 7 using the same microbubble cavity coated with poly-N-isopropylacrylamide, convert each spectra into an optical barcode, and store the spectra corresponding to each pH value and its corresponding optical barcode in a database;

[0007] S3. Using the same microbubble cavity coated with poly (N-isopropylacrylamide) as above to perform enzymatic reaction detection, the enzymatic reaction solution to be tested is introduced into the microbubble cavity, and the corresponding spectrum is collected at intervals;

[0008] S4. Use the cross-correlation function to find the spectrum in the database, extract the spectrum and its optical barcode information and pH value, and complete the pH value monitoring of the enzymatic reaction solution to be tested.

[0009] Further, the method for preparing the microbubble cavity coated with poly-N-isopropylacrylamide is:

[0010] a. dissolving poly (N-isopropylacrylamide) powder in deionized water, stirring and then standing to completely dissolve it, to obtain a polymer solution;

[0011] b. Injecting the polymer solution into the whispering gallery mode microbubble cavity with openings at both ends through a syringe pump;

[0012] c. repeatedly injecting the polymer solution into the microbubble cavity pumped out of the whispering gallery mode, so that the polymer solution is evenly coated on the inner wall of the microbubble cavity;

[0013] d. Place the coated microbubble cavity in an oven for heating to allow the polymer to solidify and coat the inner surface of the microbubble resonance cavity.

[0014] Furthermore, the poly (N-isopropylacrylamide) is sensitive to pH, and its refractive index changes after combining with hydrogen ions in an acidic solution.

[0015] An enzymatic reaction monitoring device integrated with a whispering gallery mode microbubble cavity comprises: a broadband light source, a spectrum analyzer, a tapered optical fiber, a packaging device, etc. The broadband light source is used to inject coupled light based on the whispering gallery mode microbubble microcavity. The enzymatic reaction monitoring system uses a spectrum analyzer to analyze the spectrum information and display the spectrum of the emitted light. The microbubble resonant cavity is made of a quartz capillary microtube with both ends open, and the inner surface of the microbubble cavity is coated with pH-sensitive poly-N-isopropylacrylamide, which can change its refractive index after combining with hydrogen ions in an acidic solution.

[0016] The mechanism of monitoring the solution by the microbubble cavity is as follows: a pH-sensitive polymer solution is injected into the whispering gallery mode microbubble resonant cavity with openings at both ends. The polymer solution can change its refractive index after combining with hydrogen ions in the solution to be tested.

[0017] The poly N-isopropylacrylamide powder is dissolved in deionized water, stirred for 5 minutes, and then allowed to stand for 1 hour to completely dissolve. The solution is repeatedly passed into and pumped out of the whispering gallery mode microbubble cavity to be evenly coated on the inner wall of the microbubble; then placed in an oven for heating to uniformly solidify the polymer and coat the inner surface of the microbubble resonance cavity to form a variable refractive index layer.

[0018] The microcavity is a microbubble cavity, that is, a microbubble cavity bulges in the middle of a capillary microtube.

[0019] The poly N-isopropylacrylamide solution is coated on the inner wall of the microbubble cavity after solidification.

[0020] The preparation of a whispering gallery mode microbubble cavity with openings at both ends comprises the following steps:

[0021] Select a quartz capillary microtube of appropriate length and thickness, and use a Bunsen burner to evenly heat it to remove the outer cladding of the capillary microtube. Seal one end of the quartz capillary microtube with UV glue, and use an ultraviolet lamp to cure the UV glue. Connect the other end of the quartz capillary microtube to the injection pump, start the injection pump and inject deionized water into the quartz capillary microtube at a certain speed. Put the part of the capillary microtube with the outer cladding removed into the optical fiber fusion splicer, perform arc discharge, and find that a microbubble cavity will bulge at the arc excitation point.

[0022] The method for using the whispering gallery mode microbubble cavity comprises the following steps: introducing a liquid to be tested into the whispering gallery mode microcavity; coupling light in a tapered optical fiber to a polymer refractive index variable layer on the inner wall of the microbubble cavity in the form of an evanescent field, causing total reflection to generate a whispering gallery mode; using a spectrum analyzer to collect light emitted from the polymer refractive index variable layer on the inner wall of the whispering gallery mode microbubble cavity, and converting the collected light into a resonance spectrum of the whispering gallery mode microbubble cavity; collecting a spectrum with the most similar spectral morphology from an optical barcode database, and reading out a pH value expressed by the spectrum in the database to correspond to an enzyme reaction experimental pH value detected in an experiment.

[0023] The method for establishing the optical barcode database is as follows: 100 spectra with pH values ​​ranging from 5 to 7 are collected using the same polymer-coated microbubble cavity, each spectrum is converted into an optical barcode, and then the spectrum corresponding to each pH value and its corresponding optical barcode are stored in the database.

[0024] The optical barcode database establishment and retrieval method is as follows: the same polymer-coated microbubble cavity as described above is used for enzymatic reaction detection. The enzymatic reaction solution to be tested is introduced into the microbubble cavity, and the corresponding spectrum is collected at intervals. The most similar spectrum in the database is found by using the cross-correlation function, and the spectrum, its optical barcode information and pH value are extracted to complete the pH value monitoring of the enzymatic reaction solution to be tested.

[0025] The beneficial effects of the present invention are as follows: the microcavity material is a quartz capillary microtube with openings at both ends, and its inner surface is coated with a pH-sensitive polymer. When it is exposed to a specific enzymatic reaction solution, the polymer will combine with the hydrogen ions in the solution to be tested, causing its refractive index to change. The innovation of the present invention is that the composite microcavity can conveniently and efficiently detect the pH of a specific acidic solution, and use the optical barcode technology of the whispering gallery mode to intuitively monitor biological information such as pH value. Compared with the traditional sensing method of monitoring the relative displacement between the resonance mode and the original state caused by pH changes, this method can extract additional information related to the biosensing process from the overall behavior of the WGM spectrum, and can be widely used in biochemical reaction detection, environmental protection and other fields, providing a new sensing strategy for the intelligent monitoring of biological reactions in the future.

[0026] Integrating optical barcoding technology into polymer-coated microbubble resonators has the following advantages:

[0027] 1. By overcoming the potential problem of resonant modes deviating from the swept frequency range in traditional tracking sensing methods, our method maintains a significantly improved dynamic monitoring range while maintaining high resolution.

[0028] 2. Compared with the traditional sensing method of monitoring the relative shift between the resonance mode caused by pH change and the original state, this method can extract additional information related to the biosensing process, such as the actual pH value, from the overall behavior of the WGM spectrum.

[0029] 3. The doped buffer sealed in the closed microbubble resonant cavity detected at the variable refractive index interface effectively isolates external interference and provides a suitable sensing platform for detecting harmful and volatile solutions. Therefore, the optical barcode technology of the microbubble cavity with the inner surface functionalized coating based on the whispering gallery mode proposes a new concept of reliable biological sensor with high resolution and wide dynamic range. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings are intended to provide a further understanding of the present invention, and the accompanying drawings are incorporated into the specification and constitute a part of the specification. In order to clearly explain the technical solutions in the embodiments of the present invention or the prior art, the following briefly describes the accompanying drawings required for describing the embodiments or the prior art.

[0031] Figure 1 It is a schematic diagram of the structure of the whispering gallery microbubble cavity resonator coupling device.

[0032] Figure 2 This is a structural diagram of the whispering gallery microbubble cavity resonator coupling device.

[0033] Figure 3The time-varying resonance spectrum of the polymer-coated microbubble cavity in 200 µM penicillin buffer solution corresponds to the barcode measured by the WGM resonance spectrum. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of this application.

[0035] Figure 1 and Figure 2 The main coupling device and coupling device in the present invention are shown. The coupling device is mainly composed of three parts, including a whispering gallery mode microbubble cavity 1, a tapered optical fiber 2, and a whispering gallery mode microbubble cavity packaging device 3. The device also includes a jumper connected to the tapered optical fiber, and a spectrometer and a light source respectively connected to the jumper.

[0036] The material of the whispering gallery mode microbubble cavity 1 is quartz capillary microtube, which is prepared by arc discharge of a fiber fusion splicer. The tapered optical fiber 2 is made by a fiber taper machine. While ensuring that both ends of the optical fiber remain unchanged, the coupling part with the whispering gallery microbubble cavity 1 is thinned to better couple with it. The whispering gallery mode microbubble cavity packaging device 3 is a packaging device based on a flat plate with two sections of protrusions and two rectangular frames integrated. In addition to the placement space for the whispering gallery mode microbubble cavity 1, the flat surface also reserves placement space for the tapered optical fiber 2.

[0037] First, the tapered optical fiber 2 and the jumper are welded together by a fiber fusion splicer, and the jumper is connected to the instrument required by the whispering gallery coupling system. Then, the whispering gallery mode microbubble cavity 1 is placed in the packaging device 3, and then the target solution is added to the microcavity 1 by a syringe pump. The light source is input into the tapered optical fiber through the jumper, and through precise position adjustment, the optical fiber cone is coupled with the whispering gallery microbubble resonator and the whispering gallery mode phenomenon is generated, thereby generating a target coupling spectrum at the output end of the optical fiber cone, and a spectrometer is used for observation and data collection. After that, the optical barcode technology is used to retrieve the pre-set database to find the pH value corresponding to the specific spectrum and barcode, so as to achieve high-sensitivity monitoring of the enzymatic reaction process and provide the necessary signals for subsequent biological detection or other optical detection. Figure 3 The time-varying resonance spectrum of a 200 µM penicillin buffer solution is shown along with the barcode corresponding to the measured WGM resonance spectrum. Example

[0038] The recognition of pH plays a key role in fields such as marine science, clinical analysis, and environmental science. pH biosensors based on WGM microcavities have attracted extensive research interest because they can overcome the limitations of complex equipment and biomarkers. This invention introduces a bioresponse sensing mechanism involving multimodal WGM resonances to generate optical barcodes from multimodal spectra that are uniquely related to pH. The sensing information is obtained through data analysis of the barcodes that vary with pH. Compared with traditional sensing methods that track a single mode, this new method allows for the simultaneous analysis of multimodal features in the WGM resonance spectrum, including coupling depth, linewidth, modal spacing, and other parameters. To generate WGM barcodes under different pH buffering schemes, the transmission intensity in the spectrum is converted into a one-dimensional array, where each element in the array corresponds to a rectangular area of ​​the barcode. The color directly reflects the intensity of the resonance. Each row in the barcode represents a mode in the spectrum, the linewidth reflects the linewidth of the mode, and the color reflects the coupling depth.

[0039] Compared with the traditional biosensing method of tracking a single resonance mode, WGM barcode technology combined with pH-sensitive coated microbubble cavity has the advantages of high resolution, high sensitivity and large dynamic response range without the need for additional complex components. The use of optical barcode monitoring is not limited to the proven penicillin enzymatic reaction, and the acquisition of reaction process information can also be extended to parameters such as temperature, humidity, and refractive index. The future development of this type of sensor can combine biology, photonics, and informatics to further improve the detection capability and processing speed of the sensor by combining high sensitivity and specific materials with high-speed matching algorithms.

[0040] The production of penicillinase by bacteria is the main reason why bacteria develop resistance to penicillin. It is crucial to deeply study the reaction mechanism between penicillin and penicillinase, and then develop a new type of rapid detection sensor for penicillin residues and screen new antibacterial drugs. The present invention studies the potential of a microbubble resonator with a polymer-coated inner surface as a biosensor for monitoring penicillin enzymatic reactions. The refractive index variability of the polymer coating on the inner surface of the microbubble resonator is strongly dependent on the pH of the injected buffer solution. This is because the pH change causes the polymer to swell, resulting in a change in the refractive index, thereby adjusting the position of the optical resonance spectrum. Since penicillinase can hydrolyze penicillin into penicillic acid and hydrogen ions (H + ), a buffer solution containing 200 μM (74.5 mg / ml) penicillin was injected into the cavity of the polymer-coated microbubble with a diameter of 265 μm, and the WGM resonance spectrum was collected immediately after the injection of 40 μL of a 0.2 mg / ml penicillinase solution. Figure 3The enzymatic reaction process and the time shift of the spectrum (left side) and the corresponding optical barcode (right side) are shown. Among them, the resonance wavelength remains stable for less than 2 minutes and more than 9 minutes, while the resonance wavelength is observed to be red-shifted by 5.36 nm between 2 and 9 minutes. The mode spacing, resonance wavelength, coupling depth and line width in the multimode resonance spectrum are all uniquely determined by the pH value, which are converted into the width, position and color of the line in the optical barcode. Therefore, by comparing the optical barcode from the measured multimode resonance results with the pH reference barcode database, it is observed that at the reaction time points of 4 min, 6 min and 8 min, the barcode best matches the pH values ​​of 6.6, 5.7 and 5.2 (almost consistent with the macroscopic pH measurement). The experimental results complete the monitoring of the enzymatic reaction process and the measurement of the actual pH value during the reaction in the microfluidic channel. After the injection of penicillin and penicillinase in the microbubble cavity, the refractive index of the polymer coating can be changed, and the previously calibrated optical barcode database is used to obtain the pH related to the enzymatic reaction in the microfluidic channel. The spectral response of the polymer-coated microbubble resonator was verified to monitor the H released during the enzymatic reaction. + potential in this regard.

[0041] The experimental results show that the multimode spectrum generated by the microbubble cavity makes it an excellent sensing platform, and the WGM optical barcoding technology based on tracking the overall behavior of the spectrum provides a more effective sensing method for biological reaction monitoring. Compared with the traditional method of tracking specific resonance modes, this method can monitor the dynamic direction independently of the scanning wavelength range of the spectrometer. Therefore, the dynamic range of detection is significantly enhanced, depending only on the range of pH changes allowed by the sensing material. In addition to excluding irreversible damage, the optical barcoding technology derived from the multimode spectrum provided by the WGM microcavity can be used for long-term monitoring of biological reaction processes.

[0042] Finally, it should be noted that the protection scope of this application is not limited to the applications and technical solutions disclosed in the embodiments. Any technician familiar with the technical field can make appropriate changes and substitutions according to specific application requirements and environmental conditions within the technical scope disclosed in this application. These possible changes and substitutions, including but not limited to the selection of materials, adjustment of structures, expansion of functions, and integration with other technologies, etc., should be regarded as part of the protection scope of this application.

Claims

1. A method for monitoring enzymatic reactions based on optical barcodes, characterized in that: The steps include: S1. introducing the liquid to be tested into the microbubble cavity coated with poly (N-isopropylacrylamide); coupling the light in the tapered optical fiber into the poly (N-isopropylacrylamide) layer on the inner wall of the microbubble cavity in the form of an evanescent field, causing total reflection to generate a whispering gallery mode; S2. Collect multiple spectra with pH values ​​from 5 to 7 using the same microbubble cavity coated with poly-N-isopropylacrylamide, convert each spectra into an optical barcode, and store the spectra corresponding to each pH value and its corresponding optical barcode in a database; S3. Using the same microbubble cavity coated with poly (N-isopropylacrylamide) as above to perform enzymatic reaction detection, the enzymatic reaction solution to be tested is introduced into the microbubble cavity, and the corresponding spectrum is collected at intervals; S4. Use the cross-correlation function to find the spectrum in the database, extract the spectrum and its optical barcode information and pH value, and complete the pH value monitoring of the enzymatic reaction solution to be tested.

2. The method for monitoring enzymatic reactions based on optical barcodes according to claim 1, characterized in that: The method of coating the microbubble cavity with poly-N-isopropylacrylamide is: a. dissolving poly (N-isopropylacrylamide) powder in deionized water, stirring and then standing to completely dissolve it, to obtain a polymer solution; b. Injecting the polymer solution into the whispering gallery mode microbubble cavity with openings at both ends through a syringe pump; c. repeatedly injecting the polymer solution into the microbubble cavity pumped out of the whispering gallery mode, so that the polymer solution is evenly coated on the inner wall of the microbubble cavity; d. Place the coated microbubble cavity in an oven for heating to allow the polymer to solidify and coat the inner surface of the microbubble resonance cavity.

3. The method for monitoring enzymatic reactions based on optical barcodes according to claim 1, characterized in that: The poly (N-isopropylacrylamide) is sensitive to pH, and its refractive index changes after it combines with hydrogen ions in an acidic solution.

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